1. Field of the Invention
The present invention concerns an apparatus and a method for determining the concentration of one or more chemical species in a sample by multiple luminescent emission spectrophotometric ratios, and particularly, for producing an image of the sample relating concentration of the chemical species to location in the sample.
2. Discussion of the Background
Monitoring radiative emission phenomena, such as photoluminescence (e.g., fluorescence and/or phosphorescence), as a means of determining the presence, quantity and/or concentration of a particular chemical substance in a particular sample is a well-known technique in the chemical and biochemical arts. Radiative emission phenomena occur from absorption of radiative energy (e.g., infrared, visible or ultraviolet light, x-rays, etc.) by a chemical species, which in turn, reaches an electronically excited state. In relaxing to the ground state, radiative energy is emitted by the electronically excited chemical species (e.g., infrared or visible light, etc.). Alternatively, the electronically excited chemical compound relaxes by transferring energy to a second chemical species, which in turn, emits radiative energy. In photoluminescence, relatively high-energy photons are absorbed by a compound. In turn, the electronically excited compound emits photons at a lower energy.
A chemical substance has unique radiative emission spectra that are used to characterize the substance. A photoluminescence spectrum can measure either (1) the intensity of luminescent emission at a constant emission wavelength as a function of excitation wavelength (an "excitation" spectrum) or (2) the intensity of luminescent emission at a constant excitation wavelength as a function of emission wavelength (an "emission" spectrum). For example, In an excitation spectrum, the emission intensity of a luminescent compound will exhibit characteristic maxima and minima at particular excitation wavelengths. Similarly, in an emission spectrum, the emission intensity of a luminescent compound will exhibit characteristic maxima and minima at particular emission wavelengths.
Often, the radiative emission spectra of a given chemical species are influenced by the interaction of the species with a second species present in its environment. For example, pH or the presence of metal ions can affect the energies and intensities at which a fluorescent chemical species absorbs and emits radiative energy. The changes in energies and intensities at which a given chemical species emits radiative energy can provide information concerning the second species with which it interacts.
A luminescent compound in an essentially pure solvent (e.g., deionized water) will luminesce in a characteristic manner. However, at a different pH, for example, the compound will interact to a different extent with either protons or hydroxide ions, respectively, depending on whether the solution becomes more acidic or more basic. As a result of the change in the interaction of the compound with either protons or hydroxide ions, the luminescent behavior of the compound may also change.
One can measure the fluorescence or phosphorescence intensity of a sample containing the luminescent species as either a function of excitation wavelength or emission wavelength, then take identical measurements of the luminescent compound in the presence of known concentrations of a second species which interacts with the luminescent compound and influences its luminescent behavior. From solutions of known concentrations of the compounds, one can empirically determine relationships between the intensity of a luminescent compound at a given wavelength and the relative concentration of a second, luminescence-affecting species.
In a given sample, concentrations of both the luminescent compound and the second, luminescence-affecting species may be unknown or unevenly distributed. Some locations in the sample may have high concentrations of the luminescent compound, while other locations have low concentrations. If the concentration of the second species is distributed in the sample differently from the luminescent compound, the luminescence intensity alone cannot provide information concerning the concentration of the second species.
Luminescence intensity is dependent on the amount or concentration of the luminescent species, in the absence of a second species which affects its luminescence behavior. In the presence of such a second species, the luminescence intensity is-affected in a manner proportional to the concentration of the second, luminescence-affecting species. As a result (for example, when the second species directly affects the intensity of the luminescent species), the locations in the sample having high concentrations of the luminescent compound and very high concentrations of the second species may luminesce at a given wavelength with roughly the same intensity as other locations having low concentrations of the luminescent compound and very low concentrations of the second species, if the concentration of the species at the particular emission wavelength being monitored (either the free luminescent compound or the interacting luminescent compound-second species complex) is the same. On the other hand, if the intensity of the luminescent species is inversely affected by the second species, locations in the sample having high concentrations of the luminescent compound and low concentrations of the second species could show the same intensity at a particular wavelength as locations having low concentrations of the luminescent compound and high concentrations of the second species. As a result, false determinations of the concentration of the second species result from determining the intensity of the luminescent compound at one wavelength, when the concentration of the luminescent compound is not known or the concentration distribution in the sample can vary.
This problem can be overcome by choosing two wavelengths of either emission or excitation, wherein at one wavelength, the luminescent compound exhibits a relatively high intensity, but in the presence of the second, luminescence-affecting species, exhibits a relatively low intensity; and at the other wavelength, the luminescent compound exhibits a relatively low intensity, but in the presence of the second, luminescence-affecting species, exhibits a relatively high intensity. Thus, by measuring the intensity at each of two wavelengths of either emission or excitation at which the luminescence intensity shows a strong dependance on the concentration of the second, luminescence-affecting species, one can compare the two measurements and determine the extent of the influence of the second species on the luminescence of the luminescent compound.
If one divides the intensity at the first wavelength by the intensity at the second wavelength for solutions of known concentrations of the luminescent compound and varying concentrations of the second species, one obtains a ratio of intensity in which the dependence on the concentration of the luminescent compound cancels out. Thus, one can obtain characteristic data for the concentration-dependant influence of the second species independent of the concentration of the luminescent species. Accordingly, for a sample containing a luminescent compound and a second, luminescence-affecting species, the ratio of the luminescence intensity at one wavelength of either emission or excitation to the luminescence intensity at a second wavelength, when compared to the ratios at the same wavelengths of samples of the two species at known concentrations, provides information about the concentration of the second species throughout the sample which would otherwise be difficult or impossible to obtain.
By dividing the sample into a large number of detection areas and measuring the intensity ratios of the sample in each of the areas, one can produce an image which correlates the concentration of a second, luminescence-affecting species to locations in the sample. In the biochemical arts, fluorescence ratio imaging is becoming a widely used technique. For example, fluorescence ratio imaging has been successfully employed in the analysis of calcium ions in living cells (Brooker et al, Proc. Natl. Acad. Sci. USA, 87:2813-2817 (1990); Tsien et al, Cell Calcium, 11:93-109 (1990); de Erausquin et al, Proc. Natl. Acad. Sci. USA, 87:8017-8021 (1990); DeBernardi et al, Proc. Natl. Acad. Sci. USA, 88:9257-9261 (1991); Zhang et al, J. Cell Biol., 114:155-167 (1991)) .
The general procedure in fluorescence ratio imaging (or "fluorescence ratioing") is to first measure the fluorescence intensity of a subject at two distinct wavelengths of either emission or excitation radiation, determine the ratio of the intensity at one wavelength to the intensity at the other wavelength for each location or "point" in the sample, and then print a subsequent two-dimensional image of the sample having characteristics of a third dimension (e.g., color) as a function of the value of the ratio (the ratio image). As described above, the ratio image provides information about the concentration of the interacting species (e.g., calcium) at various locations in the subject (e.g., living cells). The advantage of the two wavelength-two image approach on the same sample is that the ratio of the fluorescence intensities of the two images is purely a function of calcium ion, independent of fluorescent dye distribution within the cell, which may be uneven. (Of course, in areas where the concentration of the luminescent species is zero, no information can be obtained concerning the second species. However, this problem also exists in methods not based on ratioing.)
The dye Fura-2 is a calcium chelator that emits quantitatively different fluorescence spectra at different excitation wavelengths as a function of the concentration of free calcium ion. In the presence of a high concentration of calcium ion, Fura-2 fluoresces brightly (at high intensity) when excited at 340 nm and dimly (at low intensity) when excited at 380 nm. In the presence of a low concentration of calcium ion, the fluorescence intensities at 340 and 380 nm are reversed (dim when excited at 340 nm and bright when excited at 380 nm).
Because Fura-2 is excited at two different wavelengths, and the fluorescence emission is monitored at the same wavelength band (generally &gt;500 nm), it is considered a "dual excitation/single emission" dye. The reversal of its fluorescence characteristics in response to calcium concentration is the key to fluorescence ratio imaging using Fura-2. A theoretical depiction of the fluorescence characteristics of Fura-2 are shown graphically in FIG. 1.
Another characteristic of Fura-2 that should be noted from FIG. 1 is that the emission of Fura-2 is the same at any calcium concentration when excited at a wavelength of 360 nm. This is the isobestic point for Fura-2, or the wavelength of excitation where fluorescence is independent of calcium concentration. This property can thus be used to determine the distribution of Fura-2 within a specimen, or to measure the amount of Fura-2 at any point or location within a sample.
Fura-2 can be unevenly distributed within a cell or sample (field) of cells. The use of a ratio image (created from the ratio of two individual images) to view calcium ion distribution within a cell mathematically eliminates the variation in spatial dye distribution, since the dye concentration appears in both the numerator and denominator of the ratio, and thus, is cancelled out. Therefore, within reasonable limits, uneven distribution of dye within a specimen does not affect the validity of calcium concentration readings, since calcium concentration is a function of only the ratio of the intensity of the fluorescence of the two respective images taken at 340 nm and 380 nm. The mathematical relationship is shown in the following equation: ##EQU1## wherein: R(Lo) =the ratio of the emission intensity at 340 nm excitation to the emission intensity at 380 nm excitation at a Ca.sup.++ concentration of zero
R(Hi) =the ratio of the emission intensity at 340 nm excitation to the emission intensity at 380 nm excitation at Ca.sup.++ saturation PA1 380(Lo) =the emission intensity at 380 nm excitation at a Ca.sup.++ concentration of zero PA1 380(Hi) =the emission intensity at 380 nm excitation at Ca.sup.++ saturation PA1 Kd =dissociation constant of the Ca.sup.2+ --Fura complex in nM PA1 R =the experimentally determined 340/380 intensity ratio
Thus, the standardization data for R(Lo), R(Hi), 380(Lo) and 380(Hi) need to be obtained by viewing Fura-2 solutions containing zero and saturating concentrations of calcium. This data is then included in the ratio calculations for construction of a standard curve relating calcium concentration to the 340/380 intensity ratio (R).
Indo-1 is another fluorescent dye that exhibits calcium concentration-dependent luminescent behavior. Indo-1 is similar to Fura-2 in that its emission response to radiant excitation energy is dependent upon calcium, but Indo-1 is a single-excitation/dual-emission dye. When excited at a wavelength of 340-360 nm in the presence of a high concentration of calcium, Indo-1 emits brightly at 420 nm and emits dimly at 500 nm. In the presence of a low concentration of calcium, Indo-1 has a low fluorescence intensity at 420 nm and a high fluorescence intensity at 500 nm. The graphs for Indo-1 fluorescence look similar to those for Fura-2, except that the x-axis is changed to represent emission wavelength rather than excitation wavelength. A theoretical; depiction of the fluorescence characteristics of Indo-1 are shown graphically in FIG. 2.
By monitoring the fluorescence ratio image as a function of time of a sample containing either Fura-2 or Indo-1, one can analyze time-dependent phenomena concerning the chemical species of interest (e.g., the movement of Ca.sup.2+ across cell membranes or in response to certain biochemical stimuli). Hence, fluorescence ratio imaging has become a useful technique for monitoring the amount of a chemical species which exhibits concentration-dependent effects on the luminescence of a luminescent compound.
Imaging more than one emission ratio would permit one to draw a direct relationship between the luminescence-affecting species being monitored. By imaging essentially simultaneous multiple emission ratios on the same sample, a built-in control is provided, and the variable factors which differ between samples and the errors which inevitably occur in conducting non-concurrent experiments can be eliminated.
However, one who wishes to monitor more than one emission ratio in the same sample or substrate has been unable to do so using prior technology. Prior to the present invention, the state of available instrumentation permitted only the monitoring of a single emission intensity ratio, due to limitations with regard to the number of luminescent emissions which could accurately be monitored by the same instrument (limited to a maximum of 2).
Attempts to monitor more than one ratio using prior technology have had to rely on techniques such as employing multiple neutral density filters on a filter wheel positioned between the sample and the detector, or alternatively, changing the source of excitation energy either by multiple filters or by actually changing the source itself. These approaches introduce fatal errors, because the sample mount either had to be-removed in order to change the appropriate piece of equipment, or if automatic changers were used, vibrations sufficient to jar the sample mount and alter the visual field resulted. Furthermore, use of multiple filters or multiple excitation energy sources in conjunction with a detector having the capability of monitoring only two emission phenomena limits the range of sensitivity for additional sets of emissions.
Furthermore, the maximum speed with which the prior instrument can switch back and forth between emission measurements at each of the wavelengths being monitored is about three switches per second (i.e., the prior instrument is limited to about three fluorescence measurements per second). Even with these limitations, the prior instrument is useful for monitoring phenomena associated with a single luminescence ratio which occur over the course of from several seconds to several hours or more.
Prior to the present invention, to conduct multiple emission ratioing, one would have to conduct separate experiments for each fluorescent substance to be monitored, because the filter controlling the wavelength of excitation light must be changed to monitor the second emission. Alternatively, even if one is able to employ a source of four wavelengths of radiation, one can monitor only two emission phenomena using prior technology, since prior detectors are limited to monitoring only two emissions without having to. recalibrate the detector for reliable measurements of additional emissions. Therefore, one would have to recalibrate the detector for the emissions corresponding to the second fluorescent substance after measuring the two emissions of the first fluorescent substance, in order to monitor the ratio for the second fluorescent substance.
For example, de Erausquin et al (Proc. Natl. Acad. Sci USA, 87:8017-8021 (1990)) determined, for the same sample of cells, calcium concentration by fluorescence ratio imaging and cell viability by qualitative propidium iodide fluorescence. However, after taking the measurements for producing the calcium concentration fluorescence ratio image, de Erausquin et al required about 1 h. 45 min. to take the qualitative propidium iodide fluorescence measurements. It should also be noted that qualitative propidium iodide fluorescence to test cell viability does not involve ratioing, but rather, merely involves detection of propidium iodide fluorescence in the cell nucleus.
In any case, the procedures for imaging more than one luminescent ratio are time-consuming using prior instrumentation, and would prevent one from being able to monitor phenomena which occur on a time scale of less than about an hour or two.
If the procedures are carried out flawlessly, changing filters on a typical device for monitoring a fluorescence ratio (e.g., an inverted microscope) takes at least 5-10 minutes, and recalibrating a typical single-ratio detector takes about 10-15 minutes. Attempting to conduct multiple emission ratioing on the same sample prevents one from monitoring the concentration of a given substance with respect to time for phenomena which occur on a time scale of from several seconds to several minutes. Thus, the possibility of simultaneously monitoring many important functions of cellular physiology and biochemical behavior is precluded using prior technology.